We have shown that administration of estradiol-17 beta to male Xenopus laevis induces the mRNA coding for the estrogen receptor and activates transcription of the vitellogenin and serum retinol binding protein (RBP) genes. Using our cloned Xenopus estrogen receptor (xER) we will characterize in detail the estrogen response elements (EREs) in these genes and the domains in the xER which are responsible for DNA binding, hormone binding and transcription activation. We shall determine whether a putative vitellogenin ERE and a conserved sequence are sufficient to confer efficient estrogen inducibility on a heterologous promoter transfected into our liver cell cultures. Constructs containing mutated EREs will be analyzed by gel mobility shift assays, DNase I footprinting and transfection in order to precisely define the DNA sequences required for xER binding and for activation of gene transcription in vivo. Analysis of deleted and mutated forms of cloned xER will be used to define the DNA and hormone binding domains and to identify other regions of the xER required for transcription activation. The interaction in vitro of the mutated xER DNA binding domain with natural and mutated EREs and transfection of constructs coding for mutated receptors into receptorless cells will be used to determine whether changes in affinity for the ERE correlate well with the ability to activate gene transcription in vitro. The insensitivity to antiestrogens of xER mRNA induction and the remarkable persistence of RBP mRNA induction following estrogen withdrawal suggest that regulation of these genes involves novel control mechanisms. We shall analyze the 5' flanking regions of the xER and RBP genes to see if they contain consensus EREs, and identify any new types of DNA sequences required for induction of their mRNAs. In order to identify silencer, activator and tissue specific expression sequences in the vitellogenin promoter we will use sequence comparison with the RBP and xER promoters, and transfection of constructs containing deleted and mutated vitellogenin 5' flanking regions into liver cells. Our transcription extract from estrogen stimulated Xenopus liver will be used to achieve preferential transcription of the vitellogenin promoter, to isolate and characterize the xER- promoter transcription complex, and to identify transcription factors required for xER activation of vitellogenin gene transcription.